Inhibitor of NF-KB activation

a nf-kb and activation technology, applied in the field of new proteins, can solve the problems i-b phosphorylation is still uncertain, and the effect of tnf-induced cell killing

Inactive Publication Date: 2010-06-29
YEDA RES & DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0072](i) A pharmaceutical composition for modulating the TRA2F / NF-κB modulated / mediated effect on cells comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding NAP, or an isoform, variant, active fragment or analog thereof, according to the invention.
[0073](ii) A pharmaceutical composition for modulating the TRAF2 / NF-κB modulated / mediated effect on cells comprising as active ingredient, an antisense nucleic acid for the NAP mRNA sequence according to the invention.

Problems solved by technology

However, the mechanisms by which TRAF2, which by itself does not possess any enzymatic activity, triggers the phosphorylation of I-κB is still uncertain.
However, excess TNF can be deleterious and as such may play a major pathogenic role in a number of pathological states such as septic shock, anorexia, rheumatic diseases, inflammation and graft-vs-host reactions.
In such cases the TNF-induced cell killing has a deleterious effect.
However, the FAS / APO1 receptor may mediate deleterious effects, for example, uncontrolled killing of tissue which is observed in certain diseases such as acute hepatitis that is accompanied by the destruction of liver cells.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning

[0250]A cDNA library prepared from B-cells (Surfee et al., 1993) was screened for proteins that associate with NEMO, using the two hybrid technique as described in Boldin et al., (1996) and below. The cDNAs of this library were inserted into the XhoI site of the pACT based vector pSE1107 in fusion with the GAL4 activation domain. Yeast strain HF7c strain was used as the host strain for the purposes of transformation and screening with the two-hybrid assay. This strain carries the auxotrophic markers trp1 and leu2, and therefore cannot grow in minimal synthetic medium that lacks tryptophan and leucine, unless they also bear a plasmid carrying the wild-type versions of the genes TRP1 and LEU2. The HF7c strain also carries deletion mutations in its GAL4 and GAL80 genes (gal4-542 and ga180-538, respectively) and carries the lacZ reporter gene, fused to three copies of the GAL4 17-mer consensus sequence and the TATA portion of the CYC1 promoter in its genotype. The GA4 17-mers are...

example 2

Sequencing New Clones

[0255]Cloned cDNAs obtained above in Example 1 were purified, amplified in E. coli, and the DNA obtained therefrom was subjected to sequence analysis using an ABI automatic sequencer. The cDNA sequences of clone 10 and of clone compl. 10 are shown in FIGS. 1 and 2 respectively. The amino acid sequence of the NAP polypeptide, as deduced from the cDNA sequence of clone compl. 10, is shown in FIG. 3.

example 3

Expression of Cloned cDNAs and Interaction Between the Expressed Proteins

[0256]HeLa-Bujard cells were transfected with NEMO tagged with FLAG octapeptide sequence in pUHD10-3 based expression vector and constructs containing the open reading frame (ORF) of the selected clones fused to the Hemagglutinin (HA) (sequence encoding FLAG was introduced just in front of the site for insertion of NEMO) epitope. The cells were then grown for 24 hrs. in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% calf serum with added 35S-Methionine and 35S-Cysteine. At the end of that incubation time, cells were lysed in radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, and 1 mM EDTA; 1 ml / 5×105 cells), and the lysate was precleared by incubation with irrelevant rabbit antiserum and Protein G-Sepharose beads (Pharmacia, Sweden). Immunoprecipitation was performed by incubating aliquots of the lysate with anti-FLAG (purchased from Eastman Ko...

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Abstract

The present invention relates to a protein, designated NAP (Nemo Associated Protein), that was discovered to inhibit the activation of transcription factor NF-kB by various signals that are important in inflammatory and immune processes. The DNA and the recombinant production of this protein is also disclosed as well as methods of using the protein or encoding DNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This is a continuation-in-part of application Ser. No. 09 / 646,403, filed Sep. 18, 2000, which is a 371 national stage application of International PCT application PCT / IL99 / 00158, filed Mar. 18, 1999, the contents of which are herein incorporated entirely by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention is concerned with a novel protein that inhibits the activation of transcription factor NF-κB by various signals that are important in inflammatory and immune processes. More particularly the invention relates to a protein herein designated NAP (Nemo Associated Protein), its recombinant production and its use.[0004]2. Description of the Related Art[0005]The Tumor Necrosis Factor / Nerve Growth Factor (TNF / NGF) receptor superfamily is defined by structural homology between the extracellular domains of its members Bazan, (1993). In general, with the exception of two receptors, the p55 TNF recepto...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K14/47C07K16/18
CPCC07K14/4703
Inventor WALLACH, DAVIDKOVALENKO, ANDREICANTARELLA, GIUSEPPINA
Owner YEDA RES & DEV CO LTD
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